The Skaggs Institute
for Chemical Biology
Insights Into Protein Chemistry and Biology From Protein Structure
E.D. Getzoff, A.S. Arvai, D.P. Barondeau, E.D. Garcin, C. Hitomi, K. Hitomi, M.D.
Kroeger, A.J. Pratt, D.S. Shin, J.L. Tubbs, T.I. Wood
investigate the interface between protein structural chemistry and biology. We focus
on proteins involved in reactive oxygen defenses and in light interactions for photoactivation
and signaling. We determine high-resolution crystallographic structures and combine
these with solution analyses via hydrogen-deuterium exchange mass spectrometry and
x-ray scattering to probe conformational and dynamic changes. On the basis of our
integrated structural results, we propose comprehensive mechanistic models that
explain how proteins function as efficient catalysts and molecular machines. We
test these hypotheses with biochemical and mutational analyses to improve understanding
of how proteins achieve and regulate their activities and to aid in applications
of this knowledge for the design of proteins and inhibitors.
This year, in our Skaggs research, we achieved advances and published articles on initiation
of neuronal disease by copper, zinc superoxide dismutase (SOD) mutations, regulation
of nitric oxide synthase (NOS) and the role of this enzyme in neuronal cell death,
structural chemistry controlling posttranslational modifications in green fluorescent
protein, and roles for active-site histidines in (6-4) photolyase, which uses light
to repair DNA damage.
Chemical Biology and Regulation of SOD And NOS
Skaggs funding significantly aids our investigations of the reactive oxygen control enzymes copper,
zinc SOD and NOS. Specifically, we analyze the structural chemistry of these enzymes
to help bridge the gap from protein structures to enzyme activities in vivo. For
SOD, we designed a zinc-free variant of the human enzyme to help test the role of
zinc binding and loss in the initiation of the fatal neurodegenerative disease familial
amyotrophic lateral sclerosis, which is linked to more than 130 different mutations
in human SOD. Our results, obtained in collaboration with J.A. Tainer, the Skaggs
Institute, support the importance of the stable SOD core structure in preventing
amyloid formation and toxic effects.
For NOS, we recently reported the biphasic coupling of neuronal NOS phosphorylation to N-methyl-D-aspartate
receptors to regulate trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazole
propionic acid receptors, synaptic plasticity, and neuronal cell death, in collaboration
with G. Rameau, Johns Hopkins School of Medicine, Baltimore, Maryland, and E.B.
Ziff, New York University, New York, New York.
The 3 human NOS isozymes offer key therapeutic targets for neurotransmission (neuronal NOS),
regulation of blood pressure (endothelial NOS), and the immune response (inducible
NOS). These highly similar, but differently regulated, isozymes all synthesize the
diatomic molecule nitric oxide, which is both a molecular signal (at low concentrations)
and a cytotoxin (at high concentrations). Our crystallographic structures delineate
the NOS oxygenase module, reductase module, and calmodulin-bound linker; characterize
their cofactor binding; and identify mechanisms for their functions in the synthesis
and regulation of nitric oxide. In collaboration with Dr. Tainer, we are using small-angle
x-ray scattering to test our assembly and mechanistic models for NOS by defining
the shapes and conformational changes of NOS domains and their assemblies.
The aims of these ongoing cross-disciplinary mutational, biochemical, and structural investigations
of NOS are to (1) determine the bases for functional domain interactions, cofactor
recognition, and tuning for electron transfer and catalysis; (2) characterize the
diverse regulatory mechanisms that differentially control the NOS isozymes; and
(3) elucidate distinguishing features for isozyme-specific inhibitors. Isozyme-specific
NOS inhibitors are sought for medicinal purposes and for advancing understanding
of basic human physiology, but present a huge challenge because of active-site conservation.
We are now making strong progress in defining the basis for isozyme-specific NOS
inhibitors (Fig. 1).
|Fig. 1. Isozyme-specific inhibition of NOS. Aminopyridine inhibitor binding to human inducible NOS (top)
induces a cascade of side-chain conformational changes leading to the opening of
a new selectivity pocket. In contrast, similar binding of the same aminopyridine
inhibitor to endothelial NOS (bottom) is prevented by bulky amino acids (circled)
distant from the active-site substrate-binding pocket above the heme.
Signal Transduction in Photoactive Proteins
We use Skaggs support to discover how living things use cofactor-protein partnerships to transduce
environmental changes into appropriate biological responses. We are examining and
testing the mechanisms of light-induced protein activities in the family of green
and red fluorescent proteins used as biological markers; in the blue-light receptor
photoactive yellow protein (PYP); and in the cryptochrome flavoproteins, which are
components of circadian clocks in animals and humans, and their structural analogs,
the photolyases that repair damaged DNA bases.
For green and red fluorescent proteins, our high-resolution crystallographic structures
and mutagenesis results reveal the detailed structural chemistry for the posttranslational
modifications these proteins can create at their active sites. This chemistry is
surprisingly powerful. In fact, we tested our understanding by modifying the active
site of green fluorescent protein to achieve radical cleavage of a carbon-carbon
bond. We are now completing analyses of differences between green and red fluorescent
proteins in their active sites and protein assemblies.
PYP is the prototype for the ubiquitous Per-Arnt-Sim (PAS) domains of proteins that mediate
intraprotein and interprotein interactions and conformational changes in response
to light, oxygen, redox potential, and small-molecule ligands. Our research indicates
how light activation of PYP breaks the dark-state hydrogen-bonding network to trigger
an allosteric T(tense)-to-R(relaxed) state conformational transition for signaling.
We plan to test our proposed signal transduction mechanism within the structurally
conserved PYP/PAS fold.
We identified and characterized the CryDASH cryptochrome protein family, which occurs in animal,
plant, and bacterial species (in contrast with classic animal- and plant-specific
cryptochromes). Cryptochromes share with the homologous light-activated DNA repair
photolyases not only the overall protein fold but also the redox-active FAD cofactor
bound in an unusual U-shaped conformation and the surrounding positive electrostatic
surface consistent with a function in DNA binding. Through structural and functional
studies of diverse members of the cryptochrome/photolyase families, we are deciphering
how their similarities and differences direct the same cofactor and protein fold
to produce different biological responses to light.
Barondeau, D.P., Kassmann, C.J., Tainer, J.A., Getzoff, E.D. The case of the missing ring: radical cleavage of a carbon-carbon bond and implications
for GFP chromophore biosynthesis. J. Am. Chem. Soc. 129:3118, 2007.
Hill, N.J., Stotland, A., Solomon, M., Secrest, P., Getzoff, E., Sarvetnick, N. Resistance of the target islet tissue to autoimmune destruction contributes to genetic
susceptibility in type 1 diabetes. Biol. Direct 2:5, 2007.
Rameau, G.A., Tukey, D.S., Garcin-Hosfield, E.D., Titcombe, R.F., Misra, C., Khatri, L., Getzoff, E.D., Ziff, E.B. Biphasic coupling of neuronal nitric oxide synthase phosphorylation to the NMDA receptor regulates AMPA receptor trafficking and neuronal cell death. J. Neurosci.
Roberts, B.R., Tainer, J.A., Getzoff, E.D., Malencik, D.A., Anderson, S.R., Bomben, V.C., Meyers, K.R., Karplus, P.A., Beckman, J.S. Structural characterization of zinc-deficient human superoxide dismutase and implications for
ALS. J. Mol. Biol. 373:877, 2007.
Schleicher, E., Hitomi, K., Kay, C.W., Getzoff, E.D., Todo, T., Weber, S. Electron nuclear double resonance differentiates complementary roles for active site histidines
in (6-4) photolyase. J. Biol. Chem. 282:4738, 2007.
Yamamoto, J., Tanaka, Y., Hitomi, K., Getzoff, E.D., Iwai, S. Spectroscopic studies on a novel intramolecular hydrogen bond within the (6-4) photoproduct. Nucleic
Acids Symp. Ser. (Oxf.) 79, 2007. Issue 51.